Fragmentation of Cannabinoids by Flow Injection Analysis Tandem Mass Spectrometry (FIA-MS/MS).

BACKGROUND: The analysis of plant material from Cannabis sativa L. has long been targeted on its main psychologically active metabolite, Δ9-tetrahydrocannabinol (THC). In addition to the diverse plant composition and medicinal interest in several cannabinoids, these compounds may also be related to the different characteristics of samples sold illegally. Currently, it is indisputable that other cannabinoids should also be considered in cannabis assays. Mass spectrometry has been used to identify and characterize substances in the most different scenarios, and knowing the analyte fragmentation profile is essential for characterizing samples of diverse origin. OBJECTIVE: In this work, flow injection analysis-tandem mass spectrometry with electrospray ionization (FIA-ESI-MS/MS) in positive and negative modes was used to evaluate the fragmentation profiles of eight cannabinoids commonly found in cannabis samples: THC, tetrahydrocannabinolic acid, Δ8-tetrahydrocannabinol, cannabidiol, cannabidiolic acid, cannabigerol, cannabigerolic acid and cannabinol. METHODS: By exploring the fragmentation data from mass spectrometry, the samples were classified using a chemometric model of partial least squares discriminant analysis (PLS-DA). RESULTS: When ESI in negative mode is used with adequate collision energies, it is possible to identify differences in the fragmentation of isomers. Based on that, chemometric tools were employed to classify different samples. The PLS-DA applied to FIA-ESI-MS/MS data yielded satisfactory classification. CONCLUSION: Thus, the results presented can be applied as a preliminary tool in the analysis of unknown samples, guiding more accurate investigations in terms of chemical composition. HIGHLIGHTS: This study of the cannabinoid fragmentation pattern by flow injection MS showed that cannabinoids can be distinguished by their fragmentation spectra after negative electrospray ionization. Multivariate data analysis (PLS-DA) allowed classification of different cannabis samples.

Métrica Seis Sigma em análises clínicas e a qualidade na rotina laboratorial

Objetivo: Existe uma demanda por laboratórios de análises clínicas para mostrar as qualidades de sua rotina. Assim, os laboratórios se preocupam com os principais atributos que impactam a qualidade analítica: precisão e exatidão. A métrica sigma é uma ferramenta para acessar o desempenho analítico de forma fácil e inequívoca. No entanto, os laboratórios têm dificuldade em calcular e divulgar suas qualidades sigma. Métodos: Assim, objetivamos mostrar como obter e interpretar a qualidade sigma em um laboratório de análises clínicas localizado no sul do Brasil. Nosso trabalho analisou 19 parâmetros bioquímicos e dois hematológicos em relação às qualidades sigma obtidas. Além disso, sugerimos ações e estratégias de controle de qualidade que impactam positivamente a qualidade sigma por meio do índice de qualidade. Portanto, o conhecimento da qualidade sigma permite melhores estratégias de controle de qualidade e referências para metodologias de laboratório sobre qualidade. Resultados: Diferentes métricas sigma foram encontradas de 9 a 0,2 sigma. Essas métricas indicaram ótimos desempenhos, bem como espaço para melhorias significativas, conforme indicado pelo Índice de Qualidade. Portanto, o conhecimento das métricas sigma fornece uma referência de qualidade para o laboratório e permite que você avalie a eficiência analítica. Conclusão: Com base nessas constatações, esperamos que os laboratórios calculem suas qualidades, demonstrando o alcance da qualidade sigma. Além disso, podem estabelecer estratégias de controle de qualidade com o objetivo de melhoria contínua nas análises clínicas.

Objective: There is a demand for clinical analysis laboratories to display the qualities of their routine. Thus, laboratories are concerned with the main attributes that impact analytical quality: precision and accuracy. The sigma metric is a tool to easily and unambiguously access analytical performance. However, laboratories have difficulties in calculating and disclosing their sigma qualities. Thus, we aim to show how to obtain and interpret sigma quality in a clinical analysis laboratory located in southern Brazil. Methods: Our work analyzed 19 biochemical and two hematological parameters regarding the achieved sigma qualities. In addition, we suggest quality control actions and strategies that positively impact sigma quality through the quality index. Therefore, knowledge of sigma quality allows for better quality control strategies and benchmarks for laboratory methodologies about quality. Results: Different sigma metrics were found from 9 to 0.2 sigma. These metrics indicated great performances as well as room for significant improvement as indicated by the Quality Score. Therefore, knowledge of sigma metrics provides a quality benchmark for the laboratory and allows you to assess analytical efficiency. Conclusion: Based on these findings, we hope that laboratories will calculate their qualities, demonstrating the reach of sigma quality. In addition, they can establish quality control strategies with the objective of continuous improvement in clinical analyses.

Evaluation of activity and toxicity of combining clioquinol with ciclopirox and terbinafine in alternative models of dermatophytosis.

Dermatophytosis is a superficial fungal infection that affects humans and is very common in small animals. The treatment using the most commonly used antifungals is failing, and new therapeutic alternatives are required to combat the resistance of these fungal infections. Previous studies by the group have shown that clioquinol is an important therapeutic alternative in the treatment of dermatophytosis. The object was to conduct studies of antidermatophytic activity and the irritant potential from the double and triple combinations of clioquinol, terbinafine and ciclopirox in ex vivo and in vivo alternative models. To evaluate the irritant potential of antifungal combinations, the alternative HET-CAM method (chicken egg test chorioallantoic membrane) was used. Ex vivo models were used to assess the effectiveness of antifungal combinations, using pig hooves and veterinary fur. Any possible tissue damage was to assess through in histopathology of swine ears. HET-CAM results showed that all combinations can be classified as non-irritating, corroborated by the results of the histopathological evaluation of the pig's ear skin. Only the double combinations managed to remove 100% of the colony-forming units (CFU) formed on the pig's hooves. The clioquinol + terbinafine combination and the triple combination were more effective than clioquinol + ciclopirox in eradicating the preformed biofilm in fur of veterinary origin. These results show the potential of formulations of clioquinol in combination with antifungals for use in humans and in the veterinary field to combat dermatophytosis, as an important alternative therapy, for use in the near future.

Método convencional para o diagnóstico de micoses: um estudo comparativo / Conventional method for the diagnosis of mycoses: a comparative study

Introdução: O método clássico para o diagnóstico de micoses é realizado pelo Exame Micológico Direto (EMD) e cultural, que possibilita a visualização de estruturas fúngicas vegetativas e estruturas reprodutivas, respectivamente. Essa combinação é fundamental para reduzir possíveis erros analíticos e aumentar a precisão do diagnóstico. Métodos: Com a finalidade de verificar a frequência do EMD e cultural, e comparar seus parâmetros de sensibilidade e especificidade, realizamos uma análise retrospectiva entre janeiro de 2018 e maio de 2020, de 1603 laudos micológicos oriundos de um laboratório de análises clínicas, localizado em Porto Alegre. Resultados: Após a análise dos laudos observamos que a maioria dos casos apresentaram o EMD negativo com cultura positiva (36,24%). Na sequência, 30,87% dos casos foram de amostras negativas e 25,57% dos laudos foram positivos para ambos os exames. A minoria dos casos (7,29%) apresentou o EMD positivo com cultura negativa. Conclusão: Esta análise revelou que o exame cultural é mais sensível e específico, demonstrando uma maior confiabilidade no diagnóstico. Entretanto, vale ressaltar que a realização dos exames em conjunto, além de reduzir possíveis erros analíticos, proporciona um diagnóstico melhor fundamentado. (AU)

Introduction: The classic method for the diagnosis of mycoses is performed by both direct mycological examination (DME) and culture, which allow the visualization of vegetative and reproductive fungal structures, respectively. This combination is essential to reduce possible analytical errors and increase the accuracy of the diagnosis. Methods: To assess the frequency of DME and culture, and compare their parameters of sensitivity and specificity, we performed a retrospective analysis of 1603 mycological reports produced between January 2018 and May 2020 in a clinical analysis laboratory in Porto Alegre, southern Brazil. Results: After analyzing the reports, we observed that most cases presented a negative DME and a positive culture (36.24%). Subsequently, 30.87% of the cases were negative for both tests, and 25.57% were positive for both tests. The minority of cases (7.29%) presented a positive DME and a negative culture. Conclusion: Our analysis revealed that cultural examination is more sensitive and specific, showing greater reliability in the diagnosis. However, it is noteworthy that performing the tests together, in addition to reducing possible analytical errors, provides a more consistent diagnosis. (AU)

In Vitro additive effect on griseofulvin and terbinafine combinations against multidrug-resistant dermatophytes

ABSTRACT Griseofulvin (GF) and terbinafine (TF) are commonly used drugs to treat dermatophytosis, a fungal infection of the skin. Today there is an increase in drug resistance to these antifungals which highlight the need for alternative synergistic therapies. Minimum Inhibitory Concentration (MIC) of GF and TF were determined against fungi clinical isolates from local hospitals with values ranging 0.03-2.0 µg mL-1 and 0.24-4.0 µg mL-1, respectively. A checkboard test was used to determine the combination of GF:TF which could induce an additive effect against the fungi isolates Multidrug-resistant isolates showed susceptibility after treatment with 16:2 µg mL-1 GF:TF. An MTT assay further verified that GF and TF combinations have greater additive effect against pathological and multidrug-resistant isolates than antifungals alone. Herein we disclose GF:TF combinations that could constitute as a possible new anti-dermatophyte therapy.

In vitro synergism of a water insoluble fraction of Uncaria tomentosa combined with fluconazole and terbinafine against resistant non-Candida albicans isolates.

CONTEXT: Uncaria tomentosa D.C. (Rubiaceae) has several biological activities, including activity against resistant Candida strains. The synergistic interaction with terbinafine or fluconazole can be an important alternative to overcome this resistance. OBJECTIVES: The potential synergy between a water insoluble fraction (WIF) from Uncaria tomentosa bark and the antifungals terbinafine (TRB) and fluconazole (FLZ) against non-Candida albicans resistant strains was investigated. MATERIALS AND METHODS: TRB and FLZ, alone and combined with WIF, were tested by the checkerboard procedure using the micro-dilution technique against seven isolates of Candida glabrata and C. krusei. The molecular interactions occurring outside the cell wall were evaluated by scanning electron microscopy, Fourier transform infrared (FT-IR) and differential scanning calorimetry (DSC) analysis. RESULTS: The checkerboard inhibitory assay demonstrated synergy for WIF:TRB and WIF:FLZ combinations, respectively. The best synergistic cell damage was demonstrated unequivocally for the associations of WIF and TRB (1.95:4.0 µg/mL) and WIF and FLZ (1.95:8.0 µg/mL). The comparison of the FT-IR spectra of the antifungal alone, and in combination with WIF, allows recognizing clear differences in 3000, 1600, 1400, and 700-800 cm-1 bands. Additionally, modifications on TRB and FLZ thermograms were clearly noticed after their combination with WIF. CONCLUSIONS: DSC and infrared analysis demonstrated intermolecular interactions between WIF and either TRB or FLZ. Hence, quite likely the synergistic effect is related to interaction events occurring outside the cell wall between antifungal and cat's claw proanthocyanidins. A direct action on the cell wall is suggested, without connection with the ABC efflux pump mechanism.